1. Field of the Invention
The present invention relates to a human endothelin receptor, DNA sequence encoding the receptor, an expression vector carrying the DNA sequence, a transformant comprising the expression vector, and a method for producing a human endothelin receptor from the transformant.
2. Description of the Prior Art
An endothelin receptor (ET-receptor) is a receptor for an endothelin (ET). ET-receptors derived from animals such as bovines and rats have been known. An ET is a peptide present in various tissues in animals and is known as a strong vasoconstrictor. Cloning and sequence analysis of known ET genes have revealed that the ETs comprise three kinds of isopeptides: Endothelin 1 (ET-1), Endothelin 2 (ET-2), and Endothelin 3 (ET-3). Thereafter, it has been found that these ETs are distributed in a wide variety of vascular and non-vascular tissues (Proc. Natl. Acad. Sci. U.S.A. 86, 2863-2867 (1989); Trends in Pharmacol. Sci. 10, 374-378 (1989); and Proc. Natl. Acad. cci. U.S.A. 87, 2359-2363 (1990)). ET-1 has initially been identified as a strong vasoconstrictive peptide with 21-amino-acid residues produced by porcine vascular endothelial cells (Nature, 332, 411-415 (1988)).
It has previously been shown in vivo that ET-1 and ET-2 are much more strong vasoconstrictors than ET-3, whereas the three ET isopeptides are roughly equipotent in producing the transient vasodilation.
As described above, the analysis of nucleic acid sequences of ETs has revealed that various kinds of ET isopeptides exist. These ET isopeptides are also different in their properties. Therefore, it appears that various subtypes of ET-receptors exist. The existence of various subtypes of ET-receptors has been proved by the radioactive ligand binding studies of Watanabe, H. et al. (Biochem. Biophys. Res. Commun., 161, 1252-1259 (1989)), and Martin, E. R. et al. (J. Biol. Chem. 265, 14044-14049 (1990)). These studies indicate the existence of, at least, two kinds of ET-receptors. One of them has a higher affinity for ET-1 and ET-2 than for ET-3; and the other has an affinity for ET-1, ET-2, and ET-3 with no selectivity.
The ET-receptor is useful as a reagent for measuring the amount of ET or useful in screening for an antagonist of the ET-receptor so as to study agents for the circulatory system. Therefore, there is a demand for a structure analysis of the ET-receptor and effective production of the ET-receptor by means of genetic engineering using the information of this structural analysis.
The human endothelin receptor of the present invention comprises amino acid sequence from Asp at +1 to Asn at +407 shown in SEQ ID NO: 1.
The human endothelin receptor of the present invention comprises amino acid sequence from Met at xe2x88x9220 to Asn at +407 shown in SEQ ID NO: 1.
The DNA sequence of the present invention encodes the human endothelin receptor comprising amino acid sequence from Asp at +1 to Asn at +407 shown in SEQ ID NO: 1.
The human endothelin receptor of the present invention comprises amino acid sequence from Glu at +27 to Ser at +442 shown in SEQ ID NO: 2.
The human endothelin receptor of the present invention comprises amino acid sequence from Met at +1 to Ser at +442 shown in SEQ ID NO: 2.
The DNA sequence of the present invention encodes the human endothelin receptor comprising amino acid sequence from Glu at +27 to Ser at +442 shown in SEQ ID NO: 2.
The expression vector of the present invention comprises the DNA sequence encoding the human endothelin receptor having amino acid sequence from Asp at +1 to Asn at +407 shown in SEQ ID NO: 1.
The transformant of the present invention is obtained by introducing into a host cell the expression vector comprising the DNA sequence encoding the human endothelin receptor having amino acid sequence from Asp at +1 to Asn at +407 shown in SEQ ID NO: 1.
The expression vector of the present invention comprises the DNA sequence encoding the human endothelin receptor having amino acid sequence from Glu at +27 to Ser at +442 shown in SEQ ID NO: 2.
The transformant of the present invention is obtained by introducing into a host cell the expression vector comprising the DNA sequence encoding the human endothelin receptor having amino acid sequence from Glu at +27 to Ser at +442 shown in SEQ ID NO: 2.
The method for producing a human endothelin receptor of the present invention comprises culturing either one of the above-mentioned transformants and recovering a produced endothelin receptor.
Thus, the invention described herein makes possible the advantage of providing a human ET-receptor, DNA sequence encoding the ET-receptor, an expression vector carrying the DNA sequence, a transformant comprising the expression vector, and a method for producing an ET-receptor from the transformant.
This invention also provides an isolated human endothelin receptor having an affinity for endothelins 1 and 2, comprising an amino acid sequence from Asp at 1 to Asn at 407 of SEQ ID NO: 1.
This invention further provides an isolated human endothelin receptor precursor comprising an amino acid sequence from Met at xe2x88x9220 to Asn at 407 of SEQ ID NO: 1.
In one embodiment, the candidate compound is detectably labeled, and wherein the step of detecting comprises measuring the amount of label bound to sample.
In another embodiment, the sample comprises a cell expressing the endothelin receptor.
In another embodiment, the step of detecting comprises measuring a change in a current across the cell membrane.
In another embodiment, the current across the cell membrane decreases, and the candidate compound is determined to be an antagonist.
In another embodiment, ET-1 or ET-2 is provided to the cell prior to contacting the cell with the candidate compound.
In another emodiment, the cell contains a DNA molecule comprising a nucleic acid sequence from G at 545 to C at 1765 shown in SEQ ID NO: 1.
This invention further provides a method of manufacturing a pharmaceutical composition, comprising the steps of:
screening a library of candidate compounds by:
contacting a sample comprising a human endothelin receptor having an affinity for endothelins 1 and 2, comprising an amino acid sequence from Asp at 1 to Asn at 407 of SEQ ID NO: 1 with each candidate compound in the library,
detecting binding of the candidate compound to the endothelin receptor, and
identifying compounds which bind to the endothelin receptor;
selecting a target compound identified from the library; and
formulating said target compound with a pharmaceutically acceptable carrier.
In one embodiment, the candidate compound is detectably labeled, and the step of detecting comprises measuring the amount of label bound to the sample.
In another embodiment, the sample comprises a cell expressing the endothelin receptor.
In another embodiment, the step of detecting comprises measuring a change in a current across the cell membrane.
In another embodiment, the current across the cell membrane decreases, and the target compound is an antagonist.
In another embodiment, ET-1 or ET-2 is provided to the cell prior to contacting the cell with the candidate compound.
In another embodiment, the cell contains a DNA molecule comprising a nucleic acid sequence from G at 545 to C at 1765 shown in SEQ ID NO: 1.
This invention further provides a pharmaceutical composition produced by
screening a library of candidate compounds by:
contacting a sample comprising a human endothelin receptor having an affinity for endothelins 1 and 2, comprising an amino acid sequence from Asp at 1 to Asn at 407 of SEQ ID NO: 1 with each candidate compound in the library,
detecting binding of the candidate compound to the endothelin receptor, and
identifying compounds which bind to the endothelin receptor;
selecting a target compound identified from the library; and
formulating said target compound with a pharmaceutically acceptable carrier.
This invention further provides a method of modulating an endothelin receptor, comprising the steps of:
screening a library of candidate compounds by:
contacting a sample comprising a human endothelin receptor having an affinity for endothelins 1 and 2, comprising an amino acid sequence from Asp at 1 to Asn at 407 of SEQ ID NO: 1 with each candidate compound in the library,
detecting binding of the candidate compound to the endothelin receptor, and
identifying compounds which bind to the endothelin receptor;
selecting a target compound identified from the library; and
contacting the endothelin receptor with the target compound.
In one embodiment, the candidate compound is detectably labeled, and the step of detecting comprises measuring the amount of label bound to the sample.
In another embodiment, the sample comprises a cell expressing the endothelin receptor.
In another embodiment, the step of detecting comprises measuring a change in a current across the cell membrane.
In another embodiment, the current across the cell membrane decreases, and the target compound is an antagonist.
In another embodiment, ET-1 or ET-2 is provided to the cell prior to contacting the cell with the candidate compound.
In another embodiment, the cell contains a DNA molecule comprising a nucleic acid sequence from G at 545 to C at 1765 shown in SEQ ID NO: 1.
This invention further provides a method for treating a condition characterized by abnormal activity of endothelin receptors in a subject, comprising the steps of:
screening a library of candidate compounds by:
contacting a sample comprising a human endothelin receptor having an affinity for endothelins 1 and 2, comprising an amino acid sequence from Asp at 1 to Asn at 407 of SEQ ID NO: 1 with each candidate compound in the library,
detecting binding of the candidate compound to the endothelin receptor, and
identifying compounds which bind to the endothelin receptor;
selecting a target compound identified from the library; and
administering the target compound to the subject.
In one embodiment, the candidate compound is detectably labeled, and the step of detecting comprises measuring the amount of label bound to the sample.
In another embodiment, the sample comprises a cell expressing the endothelin receptor.
In another embodiment, the step of detecting comprises measuring a change in a current across the cell membrane.
In another embodiment, the current across the cell membrane decreases, the target compound is an antagonist, and the condition caused by a increased endothelin receptor activity.
In another embodiment, ET-1 or ET-2 is provided to the cell prior to contacting the cell with the candidate compound.
In another embodiment, the cell contains a DNA molecule comprising a nucleic acid sequence from G at 545 to C at 1765 shown in SEQ ID NO: 1.
This invention further provides a method of determining ET-1 or ET-2 in a sample, comprising the steps of:
contacting the sample with a human endothelin receptor having an affinity for endothelins 1 and 2, comprising an amino acid sequence from Asp at 1 to Asn at 407 of SEQ ID NO: 1; and
detecting binding of the sample to the endothelin receptor.
In one embodiment, the endothelin receptor is detectably labeled.
In another embodiment, the endothelin receptor is present on a cell membrane.
In another embodiment, the step of detecting comprises measuring a change in a current across the cell membrane.
In another embodiment, the cell contains a DNA molecule comprising a nucleic acid sequence from G at 545 to C at 1765 shown in SEQ ID NO: 1.
These and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures.